Selagibenzophenone B and Its Derivatives: SelB-1, a Dual Topoisomerase I/II Inhibitor Identified through In Vitro and In Silico Analyses

The development of multitargeted drugs represents an innovative approach to cancer treatment, aiming to enhance drug effectiveness while minimizing side effects. Herein, we sought to elucidate the inhibitory effect of selagibenzophenone B derivatives on the survival of cancer cells and dual topoisomerase I/II enzyme activity. Results demonstrated that among the compounds, SelB-1 selectively inhibited the proliferation and migration of prostate cancer cells while exhibiting minimal effects on healthy cells. Furthermore, SelB-1 showed a dual inhibitory effect on topoisomerases. Computational analyses mirrored the results from enzyme inhibition assays, demonstrating the compound’s strong binding affinity to the catalytic sites of the topoisomerases. To our surprise, SelB-1 did not induce apoptosis in prostate cancer cells; instead, it induced autophagic gene expression and lipid peroxidation while reducing GSH levels, which might be associated with ferroptotic death mechanisms. To summarize, the findings suggest that SelB-1 possesses the potential to serve as a dual topoisomerase inhibitor and can be further developed as a promising candidate for prostate cancer treatment.


■ INTRODUCTION
Natural product-based compounds are still the main source of marketed anticancer drugs.According to recent records, 247 drugs have been approved for cancer treatment by world authorities (FDA, EMA, etc.) from 1981 to 2019, and more than 50% of approved anticancer drugs are natural product derivatives or biosimilars. 1Recognizing the clinical potential inherent in these compounds, it becomes imperative to establish robust synthetic methodologies facilitating not only the synthesis of natural products but also their biomimetics.In many instances, the synthesis of derivatives is the sole means of accessing these compounds.
Plants belonging to the genus Selaginella (Selaginellaceae) are considered living fossils, boasting an estimated age of 400 million years.Within this genus, over 100 structurally diverse polyphenolic compounds have been identified, encompassing unique entities such as selaginellins and selaginpulvilins. 2 These compounds contribute to the rich pharmacological profile associated with Selaginella, further emphasizing the importance of advancing synthetic strategies to unlock the therapeutic potential of both natural products and their distinctive derivatives.
Selagibenzophenone A (SelA), an arylated benzophenone recently isolated by Liu et al., demonstrated inhibitory activity against PDE4D2 with an IC 50 of 1.04 μM in a cell-free enzymatic assay. 3Recently, we reported a modular synthesis of SelA and its derivatives, which were further evaluated for various biological effects. 2 These include inhibition of intracellular PDE4D2, cytotoxic activities toward various cancer cell lines, an inverse agonistic activity on the nuclear receptor RORγ, and an antimicrobial activity.Interestingly, neither SelA nor its derivatives revealed a discernible impact on cellular cAMP levels, a surrogate measure of PDE4 activity, in HEK293 cells.Derivatives SelA-1 and SelA-2 showed moderate cytotoxicities toward the investigated cancer cell lines (EC 50 range between 22.0 and 54.7 μM), and for SelA-2, the selectivity index increased to 4.1 for the PC3 cell line.The best results were obtained with derivatives SelA-3 and SelA-4.The former showed a promising potency (EC 50 = 17.7 μM) against HT-29 with a good selectivity index of 8.2, and the latter showed good potency (7.8 μM) against the PC3 cell line with a selectivity index of 3.5. 2In 2020, and Tan et al. reported the isolation of a compound with a structural formula corresponding to selagibenzophenone B (SelB). 4 However, we have demonstrated that authors elucidated its structure incorrectly, and in fact, the isolated compound was SelA. 5 Therefore, the biological effect previously associated with SelB must be ascribed to SelA. 4,6 this current report, we present an evaluation of synthetic SelB and two derivatives thereof (SelB-1 and SelB-2), which were prepared according to a previous protocol developed by us. 5 Subsequent to synthesis, these compounds underwent screening to assess their cytotoxic properties and selectivity across diverse cancer cell lines.Notably, SelB-1 exhibited remarkable potency, with an IC 50 below 10 μM, and demonstrated a notably high selectivity index against prostate cancer.Further, investigation into the mechanistic underpinnings of its action was conducted.SelB, while displaying moderate toxicity in colon and prostate cancer cell lines, holds promising pharmacophores for the derivatization of new potent anticancer agents due to its minimal toxicity level in healthy cell lines.
The compounds featured in this study, identified as having substantial cytotoxic potential, and compounds identified in our prior reports were additionally evaluated for their ability to inhibit topoisomerase enzymes.SelA and SelB derivatives exhibit a structural similarity to polycyclic groups found in well-known topoisomerase inhibitors like topotecan (TPT) and etoposide (ETP).Topoisomerases are evolutionarily conserved nuclear enzymes, which play vital roles in DNA replication, transcription, recombination, and chromatin remodeling. 7During these processes, DNA strands unwind, leading to the formation of the supercoiled, knotted DNA structures, and these DNA structures are relaxed by short tandemly one-strand (TOPO I) or two-strand breaks (TOPO II).The proactive role of topoisomerases in these fundamental processes is an attractive target for cancer treatment.
Consequently, researchers have developed selective inhibitors for both TOPO I and TOPO II, which have been employed in treating various cancers.−15 Although selective topoisomerase inhibitors are effective in decreasing prostate cancer cell progression, the compounds have several side effects including hematological toxicity, diarrhea, and neutropenia. 16Moreover, inhibiting one type of topoisomerase might inadvertently enhance the activity of the other, leading to drug resistance. 17,18Regarding this, dual inhibition of both TOPO I and TOPO II has emerged as a promising strategy to counter the simultaneous increase in topoisomerase activity and to prevent drug resistance.Promising examples of such dual topoisomerase inhibitors include elomotecan, aclurubicin, and TAS-103, both of which are currently undergoing clinical trials (NCT01435096, NCT03045627, NCT04254640, and NCT03181815). 19,20To ascertain the potential of compounds, which we have synthesized by using biomimetic approaches, as topoisomerase inhibitors, the inhibitory effects of the compounds on both TOPO I and TOPO II were analyzed.This comprehensive exploration aims to delineate the mechanistic attributes and therapeutic potential of the synthesized compounds in relation to their impact on cellular topoisomerase functions.

SelB Derivatives Selectively Inhibited the Survival of Prostate Cancer Cell Lines
The synthesized compounds were tested for their capability to inhibit cell proliferation in various human cancer cell lines, including MCF-7 (breast carcinoma), PC-3 (prostate carcinoma), and HT-29 (colon carcinoma), as well as in a healthy cell line, HUVEC (human umbilical vein endothelial cells), using the sulforhodamine B (SRB) assay.Initially, a 100 μM dose of the compounds was screened on all cell lines.The results indicated that, apart from SelB-2, all compounds significantly reduced the viability of HT-29 and PC-3 cells to below 50%.Importantly, none of the compounds displayed cytotoxic effects on the MCF-7 cell line.Notably, SelB-1 exhibited a remarkable inhibitory effect, suppressing the proliferation of HT-29, PC-3, and HUVEC cells by 78, 95, and 57%, respectively.Similarly, SelB reduced the viability of colon and prostate cancer cells by more than 50% (71 and 80%, respectively) while having no significant effect on healthy cells (28%) (Figure 2).
Hence, SelB-1 and SelB were chosen to determine their half-maximal inhibitory concentration (IC 50 ) values on both cancerous and noncancerous cells.Additionally, the selectivity index (SI) of these compounds was calculated by dividing the IC 50 value obtained for a noncancerous cell line by that for a specific cancer cell line.As shown in Table 1, the compounds selectively inhibited cell proliferation of the prostate cancer cells.The IC 50 value of the SelB-1 was found lower than 10 μM on the prostate cancer cells (5.9 μM), and the SI of the compound was 12.3.Furthermore, SelB-1 showed a good level of cytotoxicity and selectivity against HT-29 cells (IC 50 value of 17.6 μM, SI: 4.6).On the other hand, SelB had the highest SI index for both prostate and colon cancer cells (18.9 and 12.6, respectively).However, the IC 50 values of the compound on the cells were higher than that of SelB-1 (Figure S1 and Table 1).The main difference between the structure of the compounds was their number of hydroxyl (−OH) and methoxy (−OMe) groups.In most cases, the addition of the methoxy groups to the structure of the compounds increased the cytotoxic effect. 21Consequently, SelB-1 exhibited a stronger cytotoxic effect than SelB, while SelB-2 displayed the lowest cytotoxic effect despite having the highest number of methoxy groups compared to the others.This may be explained by the possible decrease in the cytotoxic potential of compounds after the replacement of the hydroxyl group with the methoxyl group. 2 According to the in vitro cell cytotoxicity assay, SelB-1 was selected as a lead compound due to its promising potency and high selectivity against prostate and colon cancer cell lines.Subsequently, the impact of the compound on the proliferation and migration capabilities of prostate and colon cancer cells was assessed through colony formation and wound healing assays.Notably, IC 50 and two-time IC 50 doses of SelB-1 significantly reduced the wound recovery of PC-3 cells by 58 and 59%, respectively, at the end of 48 h (Figure 3A).Similarly, the same doses of SelB-1 application significantly inhibited colony formation of PC-3 cells (Figure 3B).Additionally, IC 50 and two times IC 50 doses of SelB-1 significantly reduced the wound recovery of HT-29 cells by 10 and 56%, respectively.(Figure S2).These results suggest that SelB-1 application may effectively alter the invasiveness of prostate and colon cancer cells.

Topoisomerase I and II Enzyme Inhibition Assays
Recently, it has been implicated that the compounds synthesized from Selaginella plant species effectively hinder cancer cell growth with a minimal cytotoxic effect on healthy cell lines. 2,22,23−26 Furthermore, the expression levels of the enzymes were shown to be increased in prostate cancer patients; therefore, it is proposed to be used as a biomarker for prostate cancer progression. 27Similar to the previously isolated compounds, the SelA derivatives SelA-2, SelA-3, and SelA-4, which we synthesized previously, demonstrated significant inhibition of cancer cell viability.Furthermore, the compounds showed the most promising effect on the prostate cancer cell line (with respective EC 50 values of 43.4−7.8μM).To our surprise, even at a concentration of 10 μM, the compounds were not able to show significant inhibitory effects on the recombinant PDE4 enzyme activity.Accordingly, the potential targets of these synthesized compounds have remained undisclosed.
Targeting multiple biological molecules with one compound is a recently used approach for cancer treatment, for instance, dual topoisomerase I (TOPO I) and II (TOPO II) inhibition.Topoisomerase enzymes play a crucial role in DNA replication and transcription, and their overactivation has been linked to the proliferation of cancer cells. 28Consequently, various agents have been developed to inhibit topoisomerases, many of which contain multiple benzene rings. 29,30SelA (SelA-2, SelA-3, and SelA-4) and SelB (SelB and SelB-1) derivatives contain polycyclic groups similar to the topoisomerase inhibitors such as topotecan (TPT) and etoposide (ETP).Considering this    similarity, we hypothesized that these compounds might target topoisomerase enzymes.In order to validate this hypothesis, the TOPO I and TOPO II inhibitory effects of the compounds were analyzed by using TOPOGEN topoisomerase I (TOPO I) and II (TOPO II) enzyme assay kits.As shown in Figure 4A, SelA-3 showed the highest inhibitory activity (∼76%), surpassing the well-known TOPO I inhibitor TPT (75%).Moreover, SelA-4, SelB-1, and SelB inhibited TOPO I enzyme activity higher than 50% (51, 52, and 68%, respectively).The compounds with inhibitory effects on TOPO I were also analyzed for TOPO II.Accordingly, SelA-3 and SelB-1 inhibited TOPO II enzyme activity more effectively than the established TOPO II inhibitor ETP (89, 69, and 68%, respectively).On the other hand, SelA-4 showed a 58% inhibitory effect on TOPO II enzyme activity (Figure 4B).In summary, SelB-1, SelA-4, and SelA-3 appear to have the potential to act as a dual inhibitor of both TOPO I and II, whereas SelB might be a selective TOPO I inhibitor.

Molecular Docking and Molecular Dynamics (MD) Simulations
Following the enzyme inhibition assay, the interactions between the most potent compounds (with inhibitory activity exceeding 50%) and topoisomerases were examined using computational methods.Initially, the crystal structures of TOPO I (PDB ID 1sc7) 31 and TOPO II (PDB ID 4j3n) 32 were subjected to classical molecular dynamics (MD) simulations utilizing GROMACS 5.1.4software.The crystal structures contain DNA duplexes, and the simulations were performed by the protein−DNA complex.The simulations were conducted until the backbone root-mean-square deviation (RMSD) values of each structure reached equilibrium.TOPO I and TOPO II were subjected to 40 and 30 ns (ns) production simulations, respectively (Figure S3).The most representative protein structures representing the equilibrated protein structures were taken from the last 10 ns of the simulations.
After protein and ligand preparation, molecular docking analyses were conducted using YASARA structure software (generally use YASARA for molecular graphics and modeling with the old OpenGL graphics engine), 33 which utilizes AutoDock Vina 34 for molecular docking simulations.All the results obtained from molecular docking analyses are summarized in Table 2. Notably, all compounds were bound to the TOPO I with higher affinity than the reference compound TPT.Among these compounds, SelB showed the strongest binding to the two different regions of the TOPO I (−10.2/-10.1 kcal/mol).Additionally, SelB-1 and SelA-3 bound the protein with similar binding affinities compared to SelB (−9.9 and −9.8 kcal/mol, respectively).Similarly, in the case of TOPO II, SelB-1 and SelA-3 yielded the same binding affinities to the TOPO II (−9.1 kcal/mol).However, ETP bounded to the protein stronger than the compounds (−10.23 kcal/mol).Conversely, SelA-4 yielded the smallest binding affinity to TOPO I and II compared to other compounds (−9.1 and −8.6 kcal/mol, respectively) (Table 2).
Following the molecular docking analyses, the best docking poses were subjected to MD simulations to assess the stability of the compounds on the binding sites.Fifty ns long simulations were performed for all protein−ligand complexes, and RMSD values of the proteins in the complexes indicated that equilibrium was reached (Figures S4 and S5).Furthermore, lRMSD values for each ligand were determined around 0.1 nm or lower, which indicated that all the ligands stayed mostly in their binding sites during simulations (Figures S6 and S7).In terms of binding affinity, all the compounds remained in the binding sites stronger than the reference compounds (Table 2).Surprisingly, both poses of the SelB remained in the binding sites of TOPO I with relatively high binding energies (−10.8 and −13.6 kcal/mol) (Table 2).This suggests that SelB might be capable of binding to two different regions of TOPO I.
In addition to the binding affinity and binding free energy calculations, the interactions between the compounds and topoisomerases were visualized by Discovery Studio Client (Figures 5 and 6 and Figures S8 and S11   similar residues, and the compounds retained their hydrogen bonding and hydrophobic interactions during the simulations (Figure S9).Additionally, the binding of the SelA and SelB derivatives slightly decreased the root-mean-square fluctuation (RMSF) values of their respective binding residues compared to ligand-free TOPO I (Figure S10).However, the change in the position of two benzene rings between SelA and SelB derivatives (Figure 1) led to variations in the types of interactions.For instance, both SelA-3 and SelA-4 yielded a higher number of π−π and π−alkyl interactions but a smaller number of van der Waals interactions with TOPO I compared to the SelB and SelB-1 at the end of MD simulations (Figure 5A).Moreover, the change in the type of interaction also resulted in decreased stability of the SelA derivatives into the binding site of TOPO I compared to SelB derivatives in terms of binding free energy (Table 2).Notably, SelA-3 stayed in the binding site of the TOPO I with similar energy to the second pose of SelB and SelB-1.This similarity could be attributed to the SelA-3 forming the highest number of hydrogen bonding (5) and π−π and π−alkyl interactions (8) while showing the smallest number of van der Waals interactions (9) compared to all derivatives (Figure 5 and Figure S7).SelB-1 and the second pose of SelB showed the same number of hydrogen bonding (3), van der Waals ( 14), and π−π and π−alkyl interactions (4), and both remained in the TOPO I binding site with the same energy (Table 2, Figure 5, and Figure S8).The best pose of SelB showed a higher number of hydrogen bonding interactions (5) but smaller van der Waals interactions (9) compared to the second-best pose, which could explain the 2.8 kcal/mol lower binding energy of the best pose of SelB (Table 2 and Figure 5B).
All the compounds interacted with the DNA binding site of TOPO II, specifically with residues Gly 76, Glu 777, Tyr 821, and Arg 820 and DNA bases T 9 and A 12, both in molecular docking and MD simulations (Figure 6 and Figure S11).Furthermore, the compounds retained their interactions during the MD simulations (Figure S12).Additionally, binding of the compounds increased RMSF values of the residues comprising binding sites (Figure S13).
In contrast to TOPO I, A derivatives exhibited a stronger binding to TOPO II compared to SelB-1 in terms of binding free energy.The variation in the positions of the benzene rings likely enabled SelA-3 and SelA-4 to be well-positioned in the TOPO II binding site, resulting in a higher number of interactions between SelA derivatives and TOPO II compared to SelB-1 (Figures 1 and 5).Among the SelA derivatives, SelA-3 showed a higher number of hydrogen bonding and hydrophobic interactions with TOPO II compared to SelA-4.Conversely, SelA-4 remained in the binding site of TOPO II with higher energy than SelA-3 (0.4 kcal/mol) (Table 2).The enhanced stability of SelA-4 could be attributed to the scoring function of the PRODIGY-LIGAND server.The binding energy calculation between a ligand and a protein is not determined solely by the number of hydrogen bonds, π−π interactions, or van der Waals interactions but also considers atomic contacts between the ligand and protein within a 10.5 Å range. 35,36Regarding this, SelA-4 may have had a higher number of atomic contacts than SelA-3 within the corresponding range, leading to the highest binding free energy observed (Table 2).On the other hand, SelB-1 stayed in the binding site of TOPO II with lower energy than SelA derivatives (Table 2).Even though the compound showed a higher number of π−π and π−alkyl interactions (5) compared to SelA derivatives, the compound only interacted with 10 residues through van der Waals interactions fewer than SelA-3 and SelA-4 (which had 14 and 13 van der Waals interactions, respectively) (Figure 6B).Consequently, the difference in the number of van der Waals interactions likely accounted for the 1.3 and 1.7 kcal/mol binding energy difference between SelB-1 and SelA derivatives (Table 2).Altogether, our SelB-1, SelA-3, and SelA-4 could be dual TOPO I and II inhibitors, while SelB selectively inhibited TOPO I activity.In summary, based on in silico molecular docking and MD simulations, SelB-1, SelA-3, and SelA-4 showed interactions with the DNA binding sites of both TOPO I and II.However, the effects of the SelA and SelB derivatives on binding site of the TOPO I and II were different.While binding of the compounds stabilized the binding site of the TOPO I, the opposite effect was observed on the TOPO II binding site according to the RMSF calculations (Figures S10 and S13).In contrast, SelB interacted with two distinct sides of TOPO I.

SelB-1 Application Did Not Induce Apoptosis but Increased Autophagic Gene Expression
The previous study by Lapinskaite et al. examined the cytotoxic effects of SelA-3 and SelA-4 on both cancer and healthy cell lines. 2It was found that SelA-4 inhibited prostate cancer cell viability with an IC 50 value of 7.8 μM and a selectivity index (SI) of 3.5.Conversely, the same inhibitory effect was not observed for SelA-3 in prostate cancer cells.Herein, SelB-1 demonstrated higher cytotoxicity on cancer cells than SelA-4 in terms of both IC 50 and SI (Table 1).Consequently, the effect of SelB-1 on cell death and cell cyle arrest was investigated by flow cytometry analyses.Accordingly, SelB-1 treatment slightly increased the percentage of apoptotic cells; however, this induction was not significant compared to the control group (Figure 7A).Furthermore, SelB-1 application did not induce cell cycle arrest on prostate cancer cells (Figure S14).Hence, SelB-1 might not induce apoptosis and cell cycle arrest in prostate cancer cells.
The enzyme inhibition assays indicated that SelB-1 has the potential to act as a dual TOPO I and II inhibitor.−39 However, in the flow cytometry analyses, SelB-1 did not induce an increase in either apoptotic or necrotic cell percentages (Figure 7A).Thus, we investigated the effect of SelB-1 on autophagic cell death through gene expression analysis.As shown in Figure 7, the SelB-1 application increased the expression level of ATG-3, ATG-7, ATG-12, and BECN1 while not affecting the ATG-5 and LC-3 expression levels.Specifically, at the IC 50 dose, the compound increased the expression levels of ATG-3, ATG-7, ATG-12, and BECN1 genes by 1.3-fold, 2.3-fold, 2.4-fold, and 3.9-fold, respectively (Figure 7B).According to the findings, SelB-1 application might partially induce autophagy in prostate cancer cells.(Table 2).Altogether, our SelB-1, SelA-3, and SelA-4 could be dual TOPO I and II inhibitors, while SelB selectively inhibited TOPO I activity.In summary, based on in silico molecular docking and MD simulations, SelB-1, SelA-3, and SelA-4 showed interactions with the DNA binding sites of both TOPO I and II.In contrast, SelB interacted with two distinct sides of TOPO I.

SelB-1 Might Induce Ferroptosis in Prostate Cancer Cells
Induction of the autophagy-related genes is not only associated with autophagy but also is linked to ferroptosis. 40,41For instance, ATG7, a gene known for its function in autophagy, has been found to be involved in ferroptosis. 42ATG7 may regulate ferroptosis by altering cellular breakdown processes.Autophagy, which is regulated by ATG7, has been shown to increase ferroptosis by promoting the destruction of ferritin, a protein that regulates iron storage. 43Furthermore, AMPK has been demonstrated to phosphorylate Beclin-1, another essential protein involved in autophagy, in the context of ferroptosis.Beclin-1 can contribute to ferroptosis by binding to and diminishing the action of system xc-, an antiporter that regulates cysteine import. 44Based on the findings of the study, it was observed that the SelB-1 led to 2.3-and 3.9-fold increases in ATG-7 and Beclin-1 levels, respectively.These significant increases imply that the SelB-1 might have the potential to induce cell death through ferroptosis.Ferroptosis is iron-dependent cell death, which is distinct from apoptosis, autophagy, and necrosis.Alteration of the iron metabolism in the cells leads to a reduction in the glutathione (GSH) levels and induction of lipid peroxidation.Regarding this, GSH depletion and induction of lipid peroxidation are two signs of ferroptosis. 45,46o explore the potential ferroptotic effect of SelB-1, its impact on glutathione (GSH) levels and lipid peroxidation in prostate cancer cells was investigated.SelB-1 application altered GSH levels in the prostate cancer cells.Accordingly, two times IC 50 doses of SelB-1 decreased the GSH level of prostate cancer cells more significantly than etoposide (44 and 37%, respectively) (Figure 8A).
During ferroptosis, polyunsaturated fatty acids undergo peroxidation, ultimately resulting in the formation of malondialdehyde (MDA).Consequently, MDA serves as a marker for lipid peroxidation. 47According to the results shown in Figure 8B, two times IC 50 doses of SelB-1 induced lipid peroxidation compared to the control group (about 1.2 times).

■ CONCLUSIONS
In this study, we evaluated the anticancer potency/selectivity and the dual TOPO I/II inhibitory capacity of SelB derivatives through a combination of in vitro and computational analyses.Among the tested compounds, SelB-1 showed potent and selective antiproliferative activity against prostate cancer cells.Furthermore, the compound effectively altered the migration and colony formation capability of the prostate cancer cells.Additionally, even at low doses, SelB-1 demonstrated a dual inhibitory effect on both topoisomerase I and II enzymes.In addition to SelB-1, derivatives of SelA, specifically SelA-3 and SelA-4, were also found to be effective in inhibiting both topoisomerases.Through molecular docking and MD simulations, we determined that the compounds could exert their inhibitory effects by binding to the DNA binding sites of both TOPO I and II enzymes.Intriguingly, SelB-1 did not induce apoptotic cell death on prostate cancer cells.Instead, the compound triggered the expression of autophagic genes, increased lipid peroxidation (LPO), and reduced the level of glutathione (GSH).Consequently, further investigations are required to gain a comprehensive understanding of the compound's impact on autophagic and ferroptotic cell death pathways.Moreover, SelB-1 demonstrated a promising antiproliferative effect on colon cancer cells.However, the current study did not provide a detailed discussion about the compound's impact on colon cancer cell death.
Overall, our findings indicate that selagibenzophenones might serve as innovative and multitargeted pharmacophores for the development of potent antiprostate cancer agents.

Cell Lines and Culture Conditions
HT-29 (human colorectal adenocarcinoma cell line), MCF-7 (human breast cancer cell line), PC-3 (human prostate cancer cell line), and HUVEC (human umbilical vein endothelial cells) were cultured and maintained in DMEM containing 10% FBS and 100 U/mL penicillinstreptomycin.All cell lines were incubated at 37 °C under a saturating humidity atmosphere of 5% CO 2 and 95% air.Cells were passaged by tyripsin-EDTA when they reached to 60−70% confluency.

Cytotoxicity Assays
The effect of compounds on cell viability was determined by a sulforhodamine B (SRB) assay as described previously. 2 Briefly, all cells were seeded in 96-well plates at the concentration of 5 × 10 4 cells/well and incubated for 24 h.Then, various doses of (between 1 and 100 μM) SelB derivatives (in dimethyl sulfoxide (DMSO) (0.1%)) were applied to the seeded cells for 48 h.The percentage of viable cells was evaluated by the SRB assay.

Cell Proliferation and Cell Migration Assays
The effects of SelB-1 on cell proliferation and cell migration were determined as described previously. 48For the cell proliferation assay, PC-3 cells were seeded into 6-well plates at 1000 cells/well density and incubated for 24 h.After cell attachment, cells were treated with determined doses (IC 50 and 2-fold IC 50 ) of SelB-1 for 48 h.Then, the medium was discarded and replaced every 2−3 days until colony formation (approximately 50 cells/colony) was observed in wells.The number of colonies was determined by ImageJ software.
In the cell migration assay, both cells were seeded into 24-well plates at a 7.5 × 10 5 cells/well density and incubated for 24 h.At the end of incubation, cells were scraped in the middle of a well from up to the bottom side with a pipet tip to create a wound.Then, cells were treated with determined doses of SelB-1 (IC 50 and 2-fold IC 50 ).Cell images were taken with Launch ImageFocus 4 software with the integration of a Euromex inverted microscope at 0, 12, 24, and 48 h.The wound closure was analyzed using ImageJ software.

Topoisomerase I and II Enzyme Inhibition Assays
The topoisomerase I inhibitory properties of the SelA and SelB derivatives were evaluated by a TopoGEN topoisomerase I inhibition assay kit (TG1015-3A).Briefly, a topoisomerase I enzyme (2 U) was preincubated with or without compounds at 37 °C for 30 min in the reaction buffer (1 mM Tris−HCl pH 7.9, 0.1 mM EDTA, 0.015 M NaCl, 0.1% bovine serum albumin, 0.01 mM spermidine, and 5% glycerol).After incubation, the reaction volume was completed to 20 μL by the addition of 250 ng of a pHOT1 plasmid.The reaction mixture was incubated at 37 °C for 30 min.Then, the reaction was stopped by the addition of 4 μL of a stop solution (0.125% bromophenol blue, 25% glycerol, and 5% sarkosyl).
A TopoGEN topoisomerase II inhibition assay kit (TG1001-3A) was used to detect the effect of the compounds on topoisomerase II enzyme activity.Accordingly, 250 ng of the pHOT1 plasmid and the topoisomerase II enzyme (10 U) were incubated with or without compounds at 37 °C for 30 min in the reaction buffer (0.05 M Tris-Cl, pH 8.0, 0.15 M NaCl, 10 mM MgCl 2 , 2 mM ATP, 0.5 mM dithiothreitol, and 0.45 mM BSA).The reaction was stopped by the DNA samples were electrophoresed on a 1% agarose gel at 100 V for 1 h with a running buffer of TAE (Tris-acetate-EDTA).The gel was stained with 1 μg/mL SYBR Green (21414) containing the TAE buffer for 30 min.DNA bands were visualized under UV light and analyzed by Image Studio v.5.2.

In Silico Analyses
Molecular Dynamics (MD) Simulations.The protein structures of topoisomerase I (PDB ID 1sc7) 31 and topoisomerase II (PDB ID 4j3n) 32 were subjected to classical molecular dynamics simulations to obtain well-equilibrated protein structures.The 3D structure of both topoisomerase I and topoisomerase II includes DNA duplexes, with lengths of 22 and 20 base pairs, respectively.Regarding this, DNA duplexes were retained within their respective structures, and simulations were conducted involving protein−DNA complexes.The simulations were carried out utilizing GROMACS 5.1.4software 49 and the AMBER03 force field. 50Briefly, topoisomerase I and II proteins were placed in a cubic box with dimensions of 15 × 15 × 15 Å and 17 × 17 × 17 Å, respectively.Proteins were solvated with water molecules treated with a single point charge (SPC) model, 51 and sodium chloride ions were placed into the boxes to neutralize the charge of the system.Then, the system was subsequently energyminimized.After that, 100 ps MD production simulations were carried out for each system with the constant number of particles (N), pressure (P), and temperature (T), i.e., an NPT ensemble.A constant pressure of 1 bar was applied with a coupling constant of 1.0 ps, and water molecules/ions were coupled separately to a bath at 310 K with a coupling constant of 0.1 ps.The leap-frog algorithm 52 was used for integrating the equation of motion, which was integrated at 2 fs time steps.Production runs were carried out until the root-mean-square deviation (RMSD) value of proteins reached a plateau, indicating equilibration.TOPO I and TOPO II were subjected to 40 and 30 ns production simulations, respectively.The most representative structure was obtained by a cluster tool implemented in GROMACS 5.4.1.Side-chain RMSF calculations were performed by using the rmsf tool in GROMACS 5.4.1.
At the end of molecular docking analyses, the topoisomerase− ligand complexes were subjected to 50 ns classical MD simulations, and binding free energy calculations were performed on the PRODIGY-LIGAND server. 35,36Snapshots were taken for every 1 ns of the last 10 ns of the simulations (a total of 10 structures), and they were uploaded to the server.The server calculates the binding free energy by using the following equation: where ACNN, ACCN, and ACXX are the number of atomic contacts (ACs) between nitrogen−nitrogen, carbon−nitrogen, and between all other atoms and polar hydrogens.As described in Vangone et al., the binding free energy is calculated based on the number of atomic contacts (ACs) within the distance threshold of 10.5 Å. 36 Here, binding energies were predicted for the comparison of various ligands toward the same receptors.These binding free energies were not expected to be the absolute binding free energies for the ligands.The bond lengths between the ligands and their respective binding residues were performed in VMD. 53For better observations, interacting heteroatoms (C, O, N, and S) were selected on ligand and interaction residues.Molecular Docking Simulations.Molecular docking simulations were performed by the AutoDock Vina 1.1.2program 34 implemented in the YASARA structure software. 33Briefly, the most representative protein structures obtained from the MD simulations were placed in a grid box with the dimensions of 64 Å × 64 Å × 64 Å and 71 Å × 71 Å × 71 Å for TOPO I and TOPO II, respectively.This ensured that the whole protein−DNA complex was covered for ligand docking.The spacing was arranged to 1.00 Å, and exhaustiveness was set to 20.
Twenty poses were obtained, and then, they were clustered according to their binding mode and binding affinities.The greatest number of poses (approximately 70% of the total) was observed for all the compounds in the DNA binding sites of TOPO I and II.Consequently, the DNA binding sites of TOPO I and II are identified as the primary binding sites for these compounds, with the exception of SelB binding to TOPO I.In the docking analysis, the clusters for SelB were equally distributed across two distinct sites in TOPO I. Therefore, these two sites are considered potential binding sites for TOPO I.

Annexin V and PI Assays
PC-3 cells were seeded to a 6-well cell culture plate with a concentration of 5.5 × 10 5 .After cell attachment, cells were treated with determined doses of SelB-1 (IC 50 and 2-fold IC 50 ) for 48 h.Then, cells were washed with PBS and collected with trypsin.Cells were stained with a Guava Nexin reagent and propidium iodide (PI).Then, samples were analyzed using a flow cytometer and analyzed with CPX software (Beckman Coulter FC500 system, USA).

GSH Assay and MDA Assay
GSH and MDA assays were performed to evaluate effect of the SelB-1 on ferroptotic cell death.IC 50 values of the etoposide on PC-3 cells were determined to be 26.5 μM. 55Furthermore, a 25 μM application leads to an ∼20% decrease in the GSH level of PC-3 cells after 24 h of application. 56For this reason, ETP was used as a positive control in GSH and MDA assays.The PC-3 cells were seeded to a 6-well cell culture plate with a concentration of 5.5 × 10 5 .After cell attachment, cells were treated with determined doses of SelB-1 (IC 50 and 2-fold IC 50 ) for 48 h.Total protein samples were isolated from cells by using a RIPA buffer.The concentration of total protein lysates was determined by the BCA assay.Then, 30 μL of a 0.20 M Tris buffer (20 mM EDTA, pH:8.2), 10 μL of a protein sample (10 μg/well) or GSH standards, 20 μL of 0.01 M 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and 140 μL of a methanol ACS reagent (≥99.8%) were mixed in 96-well plates.Subsequently, the plate was incubated at 25 °C for 30 min.After incubation, the absorbances (405 nm) were measured using a microplate reader (Tecan Infinite 200 PRO, Switzerland).The relative GSH concentration was calculated according to the standard curve.
For the MDA assay, PC-3 cells were seeded to a 6-well cell culture plate with a concentration of 5.5 × 10 5 .After cell attachment, cells were treated with determined doses of SelB-1 (IC 50 and 2-fold IC 50 ) for 48 h.Following this treatment, the cells were harvested in a growth medium and centrifuged.The resulting pellet was suspended in a solution containing 20% trichloroacetic acid and subsequently centrifuged again at 15,000g for 30 min.The supernatant from the centrifuged cells was collected and combined with a 0.8% thiobarbituric acid solution.This mixture was incubated at 90 °C for 1 h.After incubation, the absorbance at 565 nm was measured utilizing a microplate reader (Tecan Infinite 200 PRO, Switzerland).The MDA concentration was calculated using the below formula:

Statistical Analysis
All data were reported as means ± SEM of three independent biological replicates, and differences compared to control groups were analyzed by one-way ANOVA with Dunnett's post hoc test by using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA) (*p < 0.02, **p < 0.005, and ***p < 0.001).

■ ASSOCIATED CONTENT
* sı Supporting Information

Figure 1 .
Figure1.Compounds related to this study.SelA and its derivatives SelA-1−4 and synthetic SelB and derivatives SelB-1 and SelB-2.SelB was previously mistakenly considered as a natural product, but we proved that its structure was incorrectly elucidated.

a
SI values for each compound have been determined by dividing the IC 50 values of the healthy cell line (HUVEC) by the IC 50 value of the cancerous cell lines (HT-29, MCF-7, and PC-3).
).Specifically, in both molecular docking and MD simulations, SelB-1 and the second-best pose of SelB interacted with Arg 349, Ala 351, Lys 354, and Pro 431 residues of TOPO I (Figure 5A,B and Figure S7).Similarly, SelA derivatives, SelA-3 and SelA-4, interacted with Dc 8, Dt 9, Dc 10, Da 113, Arg 349, Ala 351, Met 428, Pro 431, and Lys 751 residues of TOPO I through hydrogen bonding or hydrophobic interactions (Figure 5A,B and Figure S8).On the other hand, the best docking pose of SelB yielded interactions with Dc 8, Lys 216, Lys 439, and Arg 449 residues of TOPO I (Figure 5A,B and Figure S8).The SelA and SelB derivatives (apart from the first pose of SelB) interacted with

Table 2 .
Binding affinities (left) and binding free energies (right) of SelA and SelB derivatives to TOPO I and II